Methods and products for increasing frataxin levels and uses thereof

ABSTRACT

Methods and products (e.g., recombinant proteins) are described for increasing frataxin expression/levels in a cell, as well as uses of such methods and products, for example for the treatment of Friedreich ataxia in a subject suffering therefrom.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT application no. PCT/CA2012/050817 filed on Nov. 16, 2012 and published in English under PCT Article 21(2), which claims the benefit, under 35 U.S.C. §119(e), of U.S. provisional application Ser. No. 61/561,440, filed on Nov. 18, 2011. All documents above are incorporated herein by reference in their entirety.

SEQUENCE LISTING

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named “16189_6_SeqList”, created on Jun. 9, 2017 having a size of ˜231 Kbytes, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to increasing frataxin expression and/or levels and uses thereof, for example for the treatment of Friedreich ataxia.

BACKGROUND OF THE INVENTION

Friedreich Ataxia

Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative and cardiac disease, is caused by a trinucleotide repeat expansion mutation in the first intron of the frataxin gene (FXN) located on chromosome 9. The mutation leads to a reduced expression of the frataxin gene. Frataxin is essential for proper functioning of mitochondria. It is involved in the removal of iron and when frataxin is reduced, the iron builds up and causes free radical damage. Nerve and muscle cells are particularly sensitive to the deleterious effects. FRDA occurs in 1 in 50 000 persons in European populations but is much more frequent in the province of Quebec in Canada, because of founder effects. Males and females are affected equally. In the classic form, FRDA symptoms appear during or before the second decade of life. It is characterized by ataxia, areflexia, loss of vibratory sense and proprioception and dysarthry (Babady et al. 2007, Cooper and Schapira 2003, Harding 1981, Lynch et al. 2002, Pandolfo 1999). Moreover, FRDA patients often have systemic involvement, with cardiomyopathy, diabetes mellitus and scoliosis. Early death can result from cardiomyopathy or associated arrhythmias (Harding 1981). Degeneration of the dorsal root ganglion cells, their ascending dorsal spinal columns and the spinocerebellar tracts results in a progressive sensory ataxia. Many patients are wheelchair bound by their third decade. Associated oculomotor problems include optic atrophy, square-wave jerks and difficulty with fixation. Importantly, cognitive abilities are relatively spared. However, many patients suffer from depression (Singh et al. 2001).

Genetic Transmission

The mutation responsible for FRDA is an unstable hyper-expansion of a GAA triplet repeat located in the first intron of the frataxin gene (Campuzano et al. 1996). In normal subjects, there are 6-34 repeats, whereas in patients there are 150 or more repeats. The patients with the shorter repeats (150-200) have milder symptoms than those with longer triplex (350 to 650). In some severely affected patients there are up to 1700 repeats. Since the frataxin gene mutation is located in an intron, it does not alter the amino acid sequence of frataxin protein. There are 2-3% of FRDA patients who have a point mutation, either a missense or a non-sense (Bidichandani, Ashizawa and Patel 1997, McCormack et al. 2000, Cossee et al. 1999). Some patients with a missense mutation have less severe symptoms because the mutated protein in still functional.

Pathological Mechanism

The pathological mechanisms have been reviewed by Pandolfo et al., (Pandolfo 2006). The repeated GAA triplets would lead to the formation of triplex in the DNA, i.e., unusual non-B DNA conformations, that decrease transcription and subsequently reduce levels of the encoded protein, frataxin (level of expression is 5 to 35% of normal; Coppola et al. 2006, Coppola et al. 2009). Frataxin is a mitochondrial matrix protein and its reduction induces an iron accumulation in the mitochondria. This iron accumulation is observed in the cardiac cells of patients and in the dentate nucleus of the brain. It is associated with oxidative damage. The reduction of frataxin leads to changes in gene expression of 185 different genes (Coppola et al. 2006, Coppola et al. 2009). Thus the reduction of frataxin has profound effects of several metabolic pathways and the correction of only one of these pathways by a drug may not be ideal.

The Frataxin Protein

Frataxin is a small protein (NCBI NM_000144.4, only 210 amino acids) that promotes the biosynthesis of heme as well as the assembly and repair of iron-sulfur clusters by delivering Fe2+ to proteins involved in these pathways It also plays a primary role in the protection against oxidative stress through its ability to catalyze the oxidation of Fe2+ to Fe3+ and to store large amounts of the metal in the form of a ferrihydrite mineral. It is processed in two steps by mitochondrial processing peptidase (MPP). MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. Two forms exist, frataxin (56-210) and frataxin (81-210), which is the main form of mature frataxin (Schoenfeld et al. 2005, Condo et al. 2006).

Several strategies have been developed for treating Friedreich ataxia. These fall generally into the following 5 categories: 1) use of antioxidants to reduce the oxidative stress caused by iron accumulation in the mitochondria; 2) use of Iron chelators to remove iron from the mitochondria; 3) use of Histone Deacetylase Inhibitors (HDACIs) to prevent DNA condensation and permit higher expression of frataxin; 4) use of molecules such as cisplatin, 3-nitroproprionnic acid (3-NP), Pentamidine or erythropoietin (EPO) to boost frataxin expression; and 5) gene therapy. However, limited success has been reported thus far for these strategies, which are mostly non-specific or more difficult to test and apply in human trials.

Thus, there remains a need for new approaches to treat Friedreich ataxia.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to inducing or increasing frataxin expression/levels in a cell, and uses thereof. In an aspect, recombinant proteins derived from a TAL (transcription activator-like) effector (also referred to as TALE) may be designed and used to specifically target the frataxin promoter to increase frataxin expression. In a further aspect, a recombinant protein comprising (a) a frataxin protein or functional fragment and/or derivative thereof; and (b) a protein transduction domain, may be designed, prepared and introduced into a cell to increase the level of frataxin protein or functional fragment and/or derivative thereof within the cell. The present invention further relates to uses of such inducing or increasing frataxin expression/levels in a cell, such as for the treatment of Friedreich ataxia.

Accordingly, in a first aspect, the present invention concerns a TAL effector based recombinant protein for increasing expression of frataxin in a cell comprising: (i) a TALE domain derived from a TAL effector protein comprising a repeat variable domain (RVD) comprising a plurality of tandem repeats monomers; (ii) a nuclear localization signal; and (ii) a transcription activation domain, wherein said RVD binds to a frataxin promoter sequence, thereby allowing expression of frataxin in said cell.

In an embodiment of the above TAL effector based recombinant protein, the monomers consist of 33 or 34 amino acid residues. In an embodiment the above RVD consist of between 6.5 and 33.5 monomers. In an embodiment, the RVD consists of 12.5, 13.5 or 14.5 monomers.

In an embodiment, the TAL effector based recombinant protein of the present invention comprises a TALE domain derived from AvrBs3, Hax2, Hax3, Hax4 or AvrXa10 TAL effector protein. In an embodiment, the TALE domain is derived from the Hax3 TAL effector protein. In an embodiment, the Hax3 TAL effector protein is from Xanthomonas campestris pv. Armoraciae.

In an embodiment of the above TAL effector based recombinant protein, the transcription activation domain is a VP64 synthetic transcription activation domain.

In an embodiment, the above-mentioned nuclear localization signal is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen.

In an embodiment of the TAL effector based recombinant protein of the present invention, the above-mentioned RVD binds to:

-   -   i) Positions 5-18 of the frataxin promoter nucleotide sequence;     -   ii) Positions 21-34 of the frataxin promoter nucleotide         sequence;     -   iii) Positions 24-37 of the frataxin promoter nucleotide         sequence;     -   iv) Positions 37-50 of the frataxin promoter nucleotide         sequence;     -   v) Positions 73-86 of the frataxin promoter nucleotide sequence;     -   vi) Positions 81-94 of the frataxin promoter nucleotide         sequence;     -   vii) Positions 92-105 of the frataxin promoter nucleotide         sequence;     -   viii) Positions 103-116 of the frataxin promoter nucleotide         sequence;     -   ix) Positions 106-119 of the frataxin promoter nucleotide         sequence;     -   x) Positions 124-137 of the frataxin promoter nucleotide         sequence;     -   xi) Positions 155-168 of the frataxin promoter nucleotide         sequence; and/or         xii) Positions 168-181 of the frataxin promoter nucleotide         sequence;

wherein the frataxin promoter nucleotide sequence (SEQ ID NO:88) is as set forth in positions 1-240 of NCBI reference number NM_000144.4.

In an embodiment, the above-mentioned RVD comprises the amino acid sequence or one or more monomer comprising: (i) HD HD HD NG NG NN NN NN NG HD NI NG NN (SEQ ID NO:2); (ii) HD HD NG NN NN NG NG NN HD NI HD NG HD (SEQ ID NO:4); (iii) NN NN NG NG NN HD NI HD NG ND HD NN NG (SEQ ID NO:6); (iv) NN HD NG NG NG NN HD NI HD NI NI NI NN (SEQ ID NO:8); (v) NN HD NI HD NN NI NI NG NI NN NG NN HD (SEQ ID NO:10); (vi) NI NN NG NN HD NG NI NI NN HD NG NN (SEQ ID NO:12); (vii) NN HD NG HD HD HD HD HD NI HD NI NN NI (SEQ ID NO:14); (viii) HD HD NG NN NI NN NN NG HD NG NI NI (SEQ ID NO:16); (ix) NN NI NNNN NG HD NG NI NI HD HD NG (SEQ ID NO:18); (x) NN HD NG HD HD HD HD HD NI HD NI NN NI (SEQ ID NO:20); (xi) NN NN HD HD NI HD HD HD NI NN NN NN NN NG (SEQ ID NO:22); or (xii) HD NN HD HD NN HD NI NN HD NI HD HD HD (SEQ ID NO:24). In another embodiment, the above-mentioned RVD comprises one or more, preferably 6 or more, more preferably 6.5 or more monomers comprising the amino acid sequence as set forth in SEQ ID NO:65.

In an embodiment, the above-mentioned cell in which frataxin expression is increased has an abnormal number of trinucleotide repeats in intron 1 of the frataxin gene. In an embodiment, the cell comprises 35 or more trinucleotide repeats. In another embodiment, the cell comprises 150 or more trinucleotide repeats. In a further embodiment the cell comprises more than 150 trinucleotide repeats. In a further embodiment, the cell comprises 200 or more repeats. In a further embodiment, the cell comprises 500 or more repeats. In a further embodiment the cell comprises 1000 or more repeats.

The TAL effector based recombinant protein of the present invention may also comprise a protein transduction domain (or cell penetrating peptide). In an embodiment, the protein transduction domain is TAT or Pep-1. In an embodiment, the protein transduction domain is TAT and comprises the sequence SGYGRKKRRQRRRC (SEQ ID NO:25). In another embodiment, the protein transduction domain is TAT and comprises the sequence YGRKKRRQRRR (SEQ ID NO: 37). In another embodiment, the protein transduction domain is TAT and comprises the sequence KKRRQRRR (SEQ ID NO: 32). In another embodiment, the protein transduction domain is Pep-1 and comprises the sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO:87), In addition or alternatively to the above-mentioned protein transduction domain, the TAL effector based recombinant protein of the present invention may be coupled to liposomes to further facilitate translocation into the cell and mitochondria.

The present invention also provides a composition comprising a TAL effector based recombinant protein as defined above and a pharmaceutically acceptable carrier.

In an embodiment, the above-mentioned TAL effector based recombinant protein or composition comprising same is for increasing frataxin expression in a cell. In an embodiment, it is for use in the treatment of Friedreich ataxia.

The present invention further concerns the use of the above-mentioned TAL effector based recombinant protein or of a composition comprising same for increasing frataxin expression in a cell.

In another aspect, the present invention provides a use of the above-mentioned TAL effector based recombinant protein or of a composition comprising same for the treatment of Friedreich ataxia.

The present invention is also concerned with the use of above-mentioned TAL effector based recombinant protein for the preparation of a medicament for the treatment of Friedreich ataxia.

In a related aspect, the present invention provides a method for increasing frataxin expression in a cell, comprising transducing said cell with the above-mentioned TAL effector based recombinant protein or composition comprising same.

The present invention also concerns a method for treating Friedreich ataxia in a subject, comprising administering to the subject the above-mentioned TAL effector based recombinant protein or composition comprising same.

The present invention also provides an isolated nucleic acid encoding the above-mentioned TAL effector based recombinant protein of the present and a vector and host cell comprising same.

In another aspect, the present invention concerns a recombinant protein comprising: a) a frataxin protein or functional fragment and/or derivative thereof; and b) a protein transduction domain. In an embodiment, the protein transduction domain is Pep-1 or Tat. In an embodiment, the protein transduction domain is TAT and comprises the sequence SGYGRKKRRQRRRC (SEQ ID NO:25). In another embodiment, the protein transduction domain is TAT and comprises the sequence YGRKKRRQRRR (SEQ ID NO: 37). In another embodiment, the protein transduction domain is TAT and comprises the sequence KKRRQRRR (SEQ ID NO: 32). In another embodiment, the protein transduction domain is Pep-1 and comprises the sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO:87).

The present invention further provides a composition comprising the above-mentioned recombinant protein together with a pharmaceutical carrier.

The present invention also relates to the use of the above-mention recombinant protein for the treatment of Friedreich ataxia or for the preparation of a medicament for the treatment of Friedreich ataxia.

The present invention also concerns a method for treating Friedreich ataxia in a subject, comprising administering to the subject the above-mentioned recombinant protein or composition comprising same.

The present invention also provides an isolated nucleic acid encoding the above-mentioned recombinant protein, vector and host cell comprising same.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 is a schematic representation of the native TAL effector (TALE) hax3 from Xanthomonas campestris pv. Armoraciae depicting the tandem repeat domain and two repeat variable residues (NG, underlined) within each repeat monomer (SEQ ID NO: 57). These di-residues determine the base recognition specificity. The four most common naturally occurring di-residues used for the construction of customized artificial TAL effectors are listed together with their proposed major base specificity. NLS, nuclear localization signal; AD, transcription activation domain of the native TAL effector (Zhang et al., Nature Biotechnology, 2011);

FIG. 2 is a schematic representation of the fluorescence reporter construct of the recombinant protein described herein: pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE. EF-1a, EF-1a promoter sequence; VP64, synthetic transcription activation domain (TAD); NLS, nuclear localization signal; 2A; 2A self cleavage peptide; EGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis post-transcriptional regulatory element.

FIG. 3 shows a schematic representation of the reporter plasmid pCR3.1-frataxin-promoter-miniCMV-mCherry described herein, comprising the frataxin promoter comprising TALE DNA recognition sites. minCMV, minimal cytomegalovirus (CMV) promoter, mCherry, fluorescence reporter gene;

FIG. 4 shows the frataxin promoter nucleic acid sequence (SEQ ID NO:88) included in the reporter construct depicted in FIG. 3.

FIG. 5 shows that when the pCR3.1-TALE-VP64-EGFP expression plasmid is transfected in 293FT cells alone, only green fluorescence (Q4, lower right quadrant) was detected in the cells by flow cytometry. (A) illustrates on the horizontal axis the forward scattered area (FSC-A), an indication of the area of the cells versus the cell granularity (SSC-A) on the vertical axis. (B) illustrates the forward scattered width on the horizontal axis versus the forward scattered area (FSC-A) on the vertical axis. (C) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter). Four quadruplexes are illustrated: Q1 contains GFP negative cells but mCherry positive cells, Q2 contains GFP positive cells and mCherry positive cells, Q3 contains GFP negative cells and mCherry negative cells and Q4 contains GFP positive cells and mCherry negative cells. (D) illustrates the intensity of GFP fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (E) illustrates the intensity of mCherry fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (F) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity on the horizontal axis versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter) on the vertical axis. (G) and (H) indicate the number of cells in each of the quadruplexes defined in panel (C). In this sample the only fluorescent cells are green fluorescent cells;

FIG. 6 shows that when the pCR3.1-frataxin-promoter-miniCMV-mCherry expression plasmid is transfected in 293FT cells alone, red fluorescence (Q1, top left quadrant) was detected in only a few cells by flow cytometry. (A) illustrates on the horizontal axis the forward scattered area (FSC-A), an indication of the area of the cells versus the cell granularity (SSC-A) on the vertical axis. (B) illustrates the forward scattered width on the horizontal axis versus the forward scattered area (FSC-A) on the vertical axis. (C) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter). Four quadruplexes are illustrated: Q1 contains GFP negative cells but mCherry positive cells, Q2 contains GFP positive cells and mCherry positive cells, Q3 contains GFP negative cells and mCherry negative cells and Q4 contains GFP positive cells and mCherry negative cells. (D) illustrates the intensity of GFP fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (E) illustrates the intensity of mCherry fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (F) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity on the horizontal axis versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter) on the vertical axis. (G) and (H) indicate the number of cells in each of the quadruplexes defined in panel (C);

FIG. 7 shows that the co-transfection of 293FT cells with pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE and pCR3.1-frataxin-promoter-miniCMV-mCherry resulted in the emission of green and red fluorescence (Q2, top right quadrant). (A) illustrates on the horizontal axis the forward scattered area (FSC-A), an indication of the area of the cells versus the cell granularity (SSC-A) on the vertical axis. (B) illustrates the forward scattered width on the horizontal axis versus the forward scattered area (FSC-A) on the vertical axis. (C) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter). Four quadruplexes are illustrated: Q1 contains GFP negative cells but mCherry positive cells, Q2 contains GFP positive cells and mCherry positive cells, Q3 contains GFP negative cells and mCherry negative cells and Q4 contains GFP positive cells and mCherry negative cells. (D) illustrates the intensity of GFP fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (E) illustrates the intensity of mCherry fluorescence intensity of the horizontal axis versus the number of cells with various intensities on the vertical axis. (F) illustrates the Green Fluorescence Protein (GFP-A) fluorescence intensity on the horizontal axis versus the mCherry fluorescence intensity (as measured with the PE-Texas red filter) on the vertical axis. (G) and (H) indicate the number of cells in each of the quadruplexes defined in panel (C);

FIG. 8 shows the sequence and activity of the TALEs described herein designed to bind to frataxin promoter sequences. Nucleic acid sequences of the RVD of Tale Nos 1-12 correspond to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 respectively. Amino acid sequences of the RVD of Tale Nos 1-12 correspond to SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 respectively;

FIG. 9 shows a diagram of a plasmid coding for a TALE_(frat/VP64) construct in the pCR3.1 plasmid according to an embodiment of the present invention;

FIG. 10 shows the quantitative PCR analysis of frataxin mRNA expression 60 hours following nucleofection of a plasmid coding for TALE_(frat/VP64) No. 8 (see Table 3) in Friedreich fibroblasts. Results were normalized with cells transfected with 3 different internal controls (HPRT1, GAPDH and 18S rRNA);

FIG. 11 shows a Western blot (A) of frataxin protein expression 60 hours following nucleofection of the plasmid coding for TALE_(frat/VP64) No. 8 (see Table 3) in Friedreich fibroblasts. In (B) frataxin expression was quantified and normalized with β-actin expression (the frataxin antibody was from Mitosciences and the β-actin antibody was purchased from Sigma Aldreich);

FIG. 12 shows increased expression of frataxin mRNA and protein in rescued YG8 fibroblasts. A. Nucleofection of the YG8R fibroblasts with a GFP plasmid shows that near of 40 to 50% of the cells were transfected and thus expressed the GFP fluorescent protein B. Nucleofection of pCR3.1 TALE_(frat/vp64)#8 significantly increased frataxin mRNA (detected by Q-RT-PCR) compared with control cells not nucleofected (CONA), control cells nucleofected alone (CONB), or control cells nucleofected with a plasmid coding for a GFP. The frataxin mRNA was normalized with different internal controls (HPRT1, GAPDH and 18S rRNA. A 1.4× increase of frataxin protein expression was observed by Western blot analysis (C) when the YG8R fibroblasts were nucleofected with pCR3.1 TALE_(frat/vp64)#8 compared to the non-nucleofected cells (CON). Frataxin protein expression was normalized using β-actin as the internal control (D). The antibodies used for this experiment are the same as for the experiments shown in FIG. 11.

FIG. 13 shows the amino acid sequences of fusion proteins 6×HIS-Tat-Tale_(frat/vp64)#8 (A, SEQ ID NO:106) and 6×HIS-Pep1-t-Tale_(frat/vp)64#8 (B, SEQ ID NO:86); HHHHHH: 6×Histidine tag. YGRKKRRQRRR (SEQ ID NO:37) corresponds to the Tat motif. KETWWETWWTEWSQPKKKRKV (SEQ ID NO:87) corresponds to the Pep1 motif. The 12 RVDs of tale #8 are shaded and bold. The 4 VP16 motifs are underlined (4XVP 16=VP64);

FIG. 14 shows the expression in E. coli (BL21) of the construct 6×His-Tat-Tale_(frat/VP64) of the present invention. A. Coomassie blue staining showing total protein with or without induction with IPTG. B. Purification on a Ni column of 6His-Tat-TALE_(frat/VP64) construct of the present invention. Lane 1: M.W; Lane 2: E. coli extract, no induction; Lane 3: E coli total extract; and Lane 4: Elution from the Ni column with 250 mM NPI;

FIG. 15 shows Western Blot detection of 6×His-Tat-TALE_(frat/VP64) using anti-6×His antibody (A) or VP16 antibody (B). A. Lane 1: soluble extract of BL21 after induction with IPTG; and Lane 2: elution from the Ni column with 250 mM NPI. B. M: Molecular weight marker; NI: BL21 cells not induced; and I: BL21 cells induced with IPTG 1 mM for 5 hours at 37° C.;

FIG. 16 shows a western blot analysis for the V5 tag. 293FT cells were transfected with plasmids coding either for frataxin fused with the V5 tag (pCR3.1-Frataxin-V5, lines 2 and 7) or for 6×His-Pep-1-frataxin fused with the V5 tag (pCR3.1-6×His-Pep-1-Frataxin-V5, lines 4, 5, 6 and 8). The proteins were extracted from the cells 36 hours later. The V5 mAb detected 3 isoforms of the frataxin protein: PP: pro-protein, PI: intermediary protein, PM: mature protein;

FIG. 17 shows the purification of 6×His-Tat-Frataxin The recombinant protein was produced in E. coli and passed through a nickel affinity column. Left lines 2 and 3 represent the proteins that when through the column without attaching to it. The column was first washed with 10 mM imidazole (lines washing 1 to 3). The protein was then eluted with 100 mM imidazole (right lines 1 to 10);

FIG. 18 shows that recombinant frataxin expressed in conditional KO cells prevents cell death. (A): cells with a conditional KO frataxin gene not transfected with Cre-recombinase. Cells in B, C, D and E were transfected with Cre-recombinase/EGFP plasmid, selected by FACS for green fluorescence and placed back in culture with either no frataxin supplement (B), with 6×His-frataxin (C), with 6×His-Tat-frataxin (D) or 6×His-Pep-1-frataxin (E). Cells cultured with 6×His-Tat-frataxin or 6×His-Pep-1-frataxin survived with cells with no frataxin or 6×His-frataxin died;

FIG. 19 shows the amino acid sequence of Hax3 from Xanthomonas campestris pv. Armoraciae (Genbank Acc. No. AAYA3359.1 and GI 66270059, SEQ ID NO:40). The N-terminal and C-terminal sequences from Hax3 comprised in the TALE construct of the present invention (pLenti-EF1-α-TALE-VP64-2A-EGFP-WPRE) are shown in bold (N-terminal) and bold/underlined (C-terminal);

FIG. 20 shows the N-terminal (A) (SEQ ID NO:41) and C-terminal (B) (SEQ ID NO:42) nucleotide sequences of Hax3 from Xanthomonas campestris pv. Armoraciae comprised in the TALE construct of the present invention (e.g., pLenti-EF1-α-TALE-VP64-2A-EGFP-WPRE and per3.1-CMV-TALE-VP64 ?);

FIG. 21 (A and B) shows the complete sequence of the PCR3.1-frataxin-promoter-miniCMV-mCherry plasmid (SEQ ID NO:43);

FIG. 22 (A to L) shows the restriction map of the PCR3.1-frataxin-promoter-miniCMV-mCherry plasmid (SEQ ID NO: 43—nucleic acid sequence of plasmid). ORF1: amino acid sequence of the mCherry reporter (SEQ ID NO: 47—see FIGS. 22B and C). ORF2: hypothetical protein (SEQ ID NO: 137—see FIGS. 22E and F). ORF3: Kanamycin-neomycin resistance protein (SEQ ID NO: 138—see FIGS. 22G and H). ORF4: beta-lactamase protein for ampicillin resistance (SEQ ID NO: 139—see FIGS. 221, J and K);

FIG. 23 shows the sequence of the proximal promoter (underlined) of human frataxin included in the PCR3.1-frataxin-promoter-miniCMV-mCherry plasmid together with linking sequences in 5′ and 3′ (SEQ ID NO:44);

FIG. 24 shows the nucleotide sequence of the miniCMV promoter (SEQ ID NO:45);

FIG. 25 shows the nucleotide (SEQ ID NO:46) and amino acid sequences (SEQ ID NO:47) of the mCherry reporter;

FIG. 26 shows the nucleotide sequence of the human elongation factor 1 alpha promoter (A) (SEQ ID NO:48) and (B) of the nucleotide (SEQ ID NO:49) and amino acid (SEQ ID NO:50) sequences of the synthetic transcription activation domain of VP64;

FIG. 27 shows the amino acid and nucleotide sequences of the self cleavage peptide 2A (SEQ ID NOs: 51 and 52) (A) and of the enhanced green fluorescent protein (EGFP, SEQ ID NOs: 53 and 54) (B);

FIG. 28 (A to K) shows the complete sequence of the pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE plasmid of the present invention (SEQ ID NO:55);

FIG. 29 (A to E) shows the sequences of the various assembly modules according to the Zang technique (Nature Biotechnology, 2011). A. NI module (SEQ ID NOs: 56 (nt) and 57 (aa)); B. NG module (SEQ ID NOs:58 (nt) and and 59 (aa)); C. HD module (SEQ ID NOs: 60 (nt) and 61 (aa)); D. NN module (SEQ ID NOs: 62 (nt) and 63 (aa)). E. consensus module (SEQ ID NOs: 64 (nt) and 65 (aa));

FIG. 30 (A and B) shows an alignment between the amino acid sequences of four backbone constructs (NN (SEQ ID NO: 129), HD (SEQ ID NO: 130), NG (SEQ ID NO: 127) and NI (SEQ ID NO: 128) backbone) of the present invention comprising the NLS (KKKRK, bold (SEQ ID NO:89)), peptide 2A fused to GFP (underlined) and the variable di-residues (NN, HD, NG and NI, bold/underlined); The consensus sequence is shown (SEQ ID NO: 66).

FIG. 31 (A to E) shows an alignment between the nucleotide sequences of four backbone constructs of the present invention (NN (SEQ ID NO: 131), HD (SEQ ID NO: 134), NG (SEQ ID NO: 133) and NI (SEQ ID NO: 132) backbone) comprising the NLS (KKKRK, bold (SEQ ID NO:89)), peptide 2A fused to EGFP (underlined); the BsmBI cloning site for cloning the TALE customized repeat region (shade) and the variable di-residues (NN, HD, NG and NI, bold/underlined);

FIG. 32 (A to E) shows the nucleic acid and amino acid sequences of the variable part (RVD) of exemplary TALEs of the present invention. A. TALE #1 (SEQ ID NOs: 67 and 68 which binds to tcccttgggtcagg (SEQ ID NO: 1) in the frataxin promoter. The repeat variable di-residues of TALE #1 are: HD, HD, HD, NG, NG, NN, NN, NN, NG, HD, NI and NG in vector NN (this combination of modules comprising variable di-residues corresponds to SEQ ID NO: 2). B. TALE #2 (SEQ ID NOs: 69 and 70) which binds to the sequence: tcctggttgcactc (SEQ ID NO: 3) in the frataxin promoter. The repeat variable di-residues of TALE #2 are: HD, HD, NG, NN, NN, NG, NG, NN, HD, NI, HD and NG (in bold, corresponding nucleic acid sequences are underlined) in vector HD (this combination of modules comprising variable di-residues corresponds to SEQ ID NO: 4). C. TALE #3 (SEQ ID NOs: 71 et 72) which binds to the sequence: tggttgcactccgt (SEQ ID NO: 5) in the frataxin promoter. The repeat variable di-residues are in TALE #3 are: NN, NN, NG, NG, NN, HD, NI, HD, NG, HD, HD and NN in vector NG (this combination of modules comprising variable di-residues corresponds to SEQ ID NO: 6). D. TALE #4 (SEQ ID NOs: 73 and 74) which reacts with the sequence: tgctttgcacaaag (SEQ ID NO: 7) in the frataxin promoter. The repeat variable di-residues in TALE #4 are: NN, HD, NG, NG, NG, NN, HD, NI, HD, NI, NI and NI (in bold, corresponding nucleic acid sequences are underlined) in vector NN (this combination of modules comprising variable di-residues corresponds to SEQ ID NO: 8). E. TALE#5 (SEQ ID NOs: 75 and 76) which reacts with the sequence: tgcacgaatagtgc (SEQ ID NO: 9) in the frataxin promoter. The repeat variable di-residues of TALE #5 are: NN, HD, NI, HD, NN, NI, NI, NG, NI, NN, NG and NN in vector HD (this combination of modules comprising variable di-residues corresponds to SEQ ID NO: 10). See Table 3 and FIG. 8 for details on other TALEs of the present invention;

FIG. 33 shows the sequence the HIS-PEP1-Frataxin construct in vector pet-16b. A. Amino acid sequence (SEQ ID NO: 78) and corresponding nucleic acid sequence (SEQ ID NO: 77) of the HIS-PEP1-frataxin recombinant protein. B. Amino acid sequence of the HIS-PEP1-frataxin recombinant protein (SEQ ID NO: 78). C. depicts the cloning sites of the 6×HIS-PEP1-frataxin in vector pet 16B (Nco I: SEQ ID NO: 135; and bamHI: SEQ ID NO: 136). D. Amino acid sequence and corresponding nucleic acid sequence (SEQ ID NO: 79) of the HIS-Tat-frataxin recombinant protein. E. Amino acid sequence (SEQ ID NO: 80) of the HIS-TAT-frataxin recombinant protein. F. depicts the cloning sites of the 6×HIS-TAT-frataxin in vector pet 16B (Nco I: SEQ ID NO: 135; and bamHI: SEQ ID NO: 136). G. Amino acid sequence and corresponding nucleic acid sequence (SEQ ID NO: 81) of the HIS-frataxin recombinant protein. H. Amino acid sequence (SEQ ID NO: 82) of the HIS-frataxin recombinant protein. I. depicts the cloning sites of HIS-frataxin in vector pet 16B (Nco I: SEQ ID NO: 135; and bamHI: SEQ ID NO: 136). 6×His tag is underlined; the PTD (PEP1(A-B) or TAT(D-E)) is shown in bold and the frataxin sequence is shaded. J and K. Nucleic acid sequence ID NO: 83) of pet-16B vector.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to inducing or increasing frataxin expression/levels in a cell, and uses thereof. In an aspect the present invention relates to the design of TAL-effector based recombinant proteins for inducing the expression of frataxin. In a further aspect, a recombinant protein comprising (a) a frataxin protein or functional fragment and/or derivative thereof; and (b) a protein transduction domain, may be designed, prepared and introduced into a cell, thereby to increase the level of frataxin protein or functional fragment and/or derivative thereof within the cell. The present invention further relates to uses of such induction or increasing frataxin expression/levels in a cell, such as for enhancing/increase expression of the frataxin protein in cells from a subject in need thereof, such as for the treatment of Friedreich ataxia.

TAL-effector based recombinant proteins (TAL Effector or TALE proteins) are naturally produced by a plant pathogen Xanthomonas sp (Boch et al. 2009; Boch and Bonas; Moscou and Bogdanove 2009). The TALEs have a highly conserved and repetitive region within the middle of the protein, consisting of tandem repeats of 33 or 34 amino acid segments (FIG. 1). These repeat monomers differ from each other mainly in amino acid positions 12 and 13 and there is strong correlation between this pair of amino acids and the corresponding nucleotide in the TALE-binding site (e.g., NI to A, HD to C, NG to T, and NN to G or A). A detailed Golden Gate PCR assembly method to produce TALEs targeting desired DNA sequences has been published (Zhang et al.) with reagents available commercially (e.g., Addgene Inc.).

In order to provide clear and consistent understanding of the terms in the instant application, the following definitions are provided.

Definitions

The articles “a,” “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps and are used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”.

The TAL effector-based recombinant proteins of the present invention are derived from naturally occurring Transcription activator-like effector (TALE, see FIG. 1) and induce the transcription of the frataxin gene and expression of the frataxin protein.

TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific activity of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain).

Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (i.e., about 20 amino acids) and is generally referred to as a “half-repeat”. Each repeat of the TAL effector feature a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze and Boch., 2010; Virulence. September-October; 1(5):428-32. doi: 10.4161/viru.1.5.12863).

Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence (see FIG. 29 and consensus module defined by SEQ ID NOs: 64 and 65). Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. The experimentally validated code between the repeat variable di-residues (RVD) sequence and target DNA base can be expressed as NI=A (module NI, SEQ ID NOs: 56 and 57), HD=C (module HD, SEQ ID NOs: 60 and 61), NG=T (NG module, SEQ ID NOs: 58 and 59), NN=G or A (NN module, SEQ ID NOs: 62 and 63), NK=G (SEQ ID NOs: 140 and 141), and NS=A, C, G, or T (SEQ ID NOs: 142 and 143—see also consensus module defined by SEQ ID NOs: 64 and 65). Target sites of TAL effectors also tend to include a T flanking the 5′ base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXa10 and AvrBs3.

Accordingly, the “TAL domain” of the TAL effector-based recombinant protein of the present invention may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. oryzicola strain BLS256 (Bogdanove et al. 2011). As used herein, the “TAL domain” in accordance with the present invention comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain (e.g., SEQ ID NOs: 41 (N-terminal) and 42 (C-terminal)) also from the naturally occurring TAL effector. It may comprise more of fewer repeats than the RVD of the naturally occurring TAL effector. The RVD domain of the TAL effector-based recombinant protein of the present invention is designed to target a given DNA sequence on the frataxin promoter based on the above code (i.e., NI=A (module NI, SEQ ID NOs: 56 and 57), HD=C (module HD, SEQ ID NOs: 60 and 61), NG=T (NG module, SEQ ID NOs: 58 and 59), NN=G or A (NN module, SEQ ID NOs: 62 and 63), NK=G (module NK, SEQ ID NOs: 140 and 141), and NS=A, C, G, or T (module NS, SEQ ID NOs: 142 and 143), consensus module: SEQ ID NOs: 64 and 65). The number of repeats (monomers or modules, see FIG. 29 and SEQ ID NOs: 64 and 65 for consensus module) and their specific sequence are selected based on the desired DNA target sequence on the frataxin promoter. For example repeats may be removed or added in order to suit a specific target sequence on the frataxin promoter. In an embodiment, the Tal-effector based recombinant protein of the present invention comprises between 6.5 and 33.5 repeats. In an embodiment, Tal-effector based recombinant protein of the present invention comprises between 8 and 33.5 repeats, preferably between 10 and 25 repeats and more preferably between 10 and 14 repeats.

Although a perfect match is preferred, a mismatch between a repeat and a target base-pair on the frataxin promoter sequence is also permitted as along as it still allows for an increase in frataxin expression. In general, TALE activity is inversely correlated with the number of mismatches. Preferably, the RVD domain of the recombinant protein of the present invention comprises 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, more preferably 2 mismatches, or less, and even more preferably no mismatch, with the corresponding target frataxin promoter sequence. Of course, the smaller the number of repeat in the RVD domain the smaller the number of mismatches tolerated. The binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, RVD domains having 25 repeats or more may be able to tolerate up to 7 mismatches.

In addition to the RVD domain, the “TALE domain” of the present invention comprises on each side of the RVD domain (i.e., on the C-terminal (e.g., SEQ ID NO: 41) and N-terminal (e.g., SEQ ID NO: 42) sides of the tandem repeats) additional sequences derived from a naturally occurring TAL effector (e.g., FIGS. 19 and 20). The length of the C-terminal and/or N-terminal sequence(s) included on each side of the RVD domain can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, to preserve the highest level of TALE activity, the first 68 amino acids on the C-terminal side of the RVD domain of the naturally occurring TAL effector is preferably included in the “TALE domain” of the recombinant protein of the present invention. Accordingly, in an embodiment, the “TALE domain” of the present invention comprises for example 1) an RVD domain derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side (e.g., FIGS. 19 and 20 and SEQ ID NOs:40, 41) of RVD domain; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side (e.g., FIGS. 19 and 20 and SEQ ID NOs:40, 42) of RVD domain.

The Tal effector-based recombinant protein of the present invention further comprises one or more (i.e, at least one) of a “transcription activation domain” or “trans-activating domain” (TAD), which contain binding sites for other proteins (e.g., transcription coregulators) and is essential for activating transcription of the target frataxin gene and expression of the frataxin protein.

Trans-activating domains (TADs) are named after their amino acid composition. These amino acids are either essential for the activity or simply the most abundant in the TAD. Transactivation by the Gal4 transcription factor is mediated by acidic amino acids, whereas hydrophobic residues in Gcn4 play a similar role. Hence, the TADs in Gal4 and Gcn4 are referred to as acidic or hydrophobic activation domains, respectively.

Nine-amino-acid transactivation domain (9aaTAD) defines a novel domain common to a large superfamily of eukaryotic transcription factors represented by Gal4, Oaf1, Leu3, Rtg3, Pho4, GIn3, Gcn4 in yeast and by p53, NFAT, NF-κB and VP16 in mammals. Prediction for 9aa TADs (for both acidic and hydrophilic transactivation domains) is available online from ExPASy™ and EMBnet™ Spain.

KIX domain of general coactivators Med15(Gal11) interacts with 9aaTAD transcription factors Gal4, Pdr1, Oaf1, Gcn4, VP16, Pho4, Msn2, Ino2 and P201. Interactions of Gal4, Pdr1 and Gcn4 with Taf9 were reported. 9aaTAD is a common transactivation domain recruits multiple general coactivators TAF9, MED15, CBP/p300 and GCN5. Accordingly, non-limiting examples of TAD that may be used in accordance with the present invention include TAD from Gal4, Pdr1, Oaf1, Gcn4, Pho4, Msn2, Ino2, P201, p53, VP16, MLL, E2A, HSF1, NF-1L6, NFAT1 and NF-kappaB. Other non-limiting examples of TAD include TAD from the SRF, TFAP2 or SP1 transcription factor, for which target sequences have been indentified in the frataxin promoter (Li et al. 2010). Of course, the choice of a TAD will depend on numerous factors including the specific type of cells in which the gene will be expressed as well as the nature of the gene. Furthermore, one can appreciate that more than one TAD may be included in a TALE construct of the present invention. In an embodiment, TAD of the recombinant portein of the present invention is VP64 which corresponds to 4 times the sequence of the VP16 TAD. In an embodiment, the TAD has the sequence GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSR (SEQ ID NO:26).

TABLE 1 Exemplary TADs from transcription factors. Annotated Peptide-KIX 9aaTAD interaction (NMR data) p53TAD1 E TFSD LWKL LSPEETFSDLWKLPE P53TAD2 D DIEQ WFTE QAMDDLMLSPDDIEQWFTEDPGPD MLL S DIMD FVLK DCGNILPSDIMDFVLKNTP E2A D LLDF SMMF PVGTDKELSDLLDFSMMFPLPVT Rtg3 E TLDF SLVT E2A homolog CREB R KILN DLSS RREILSRRPSYRKILNDLSSDAP CREBaB6 E AILA ELKK CREB-mutant binding to KIX Gli3 D DVVQ YLNS TAD homology to CREB/KIX Gal4 D DVYN YLFD Pdr1 and Oaf1 homolog Oaf1 D LFDY DFLV DLFDYDFLV Pip2 D FFDY DLLF Oaf1 homolog Pdr1 E DLYS ILWS EDLYSILWSDWY Pdr3 T DLYH TLWN Pdr1 homolog

The sequences of the annotated 9aa TADs in Table 1 above correspond, in order of appearance from top to bottom, to SEQ ID NOs:107-119. The sequences of the Peptide-KIX interaction listed on the right hand column of Table 1 above correspond, in order of appearance from top to bottom, to SEQ ID NOs:120-126 (p53 TAD1; p53TAD2, MLL, E2A, CREB, Oaf1 and Pdr1, respectively).

The Tal effector-based recombinant protein of the present invention also comprises a Nuclear Localization Signal (NLS). Accordingly, as used herein the expression “nuclear localization signal” or “NLS” refers to an amino acid sequence, which ‘tags’ a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal, which targets proteins out of the nucleus. Classical NLSs can be further classified as either monopartite or bipartite. The first NLS to be discovered was the sequence PKKKRKV (SEQ ID NO: 27) in the SV40 Large T-antigen (a monopartite NLS). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO:28), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids.

There are many other types of NLS, which are said to be “non-classical”, such as the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Mata2, the complex signals of U snRNPs as well as a recently identified class of NLSs known as PY-NLSs. Thus, any type of NLS (classical or non-classical) may be used in accordance with the present invention as long as it targets the protein of interest into the nucleus of a target cell. Preferably, the NLS is derived from the simian virus 40 large T antigen. In an embodiment, the NLS of the TAL effector based recombinant protein of the present invention has the following amino acid sequence: SPKKKRKVEAS (SEQ ID NO:29). In an embodiment the NLS has the sequence KKKRKV (SEQ ID NO:30). In an embodiment, the NLS has the sequence SPKKKRKVEASPKKKRKV (SEQ ID NO:31). In another embodiment, the NLS has the sequence KKKRK (SEQ ID NO:89).

The TAL effector-based recombinant protein of the present invention may advantageously be coupled to a protein transduction domain to ensure entry of the protein into the target cells.

In a further aspect, the present invention provides a recombinant “frataxin-protein transduction domain” protein comprising (a) a frataxin protein or functional fragment and/or derivative thereof; and (b) a protein transduction domain.

Protein transduction domains (PTD) are of various origins and allows intracellular delivery of a given therapeutic by facilitating the translocation of the protein/polypeptide into a cell membrane, organelle membrane, or vesicle membrane. PTD refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle including the mitochondria. In an embodiment, a PTD is covalently linked to the amino terminus of a recombinant protein of the present invention. In another embodiment, a PTD is covalently linked to the carboxyl terminus recombinant protein of the present invention. Exemplary protein transduction domains include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 37); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al., Cancer Gene Ther. 2002 June; 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al., Diabetes 2003; 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. Pharm. Research, 21:1248-1256, 2004); polylysine (Wender et al., PNAS, Vol. 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:33); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:34); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:35); and RQIKIWFQNRRMKWKK (SEQ ID NO:36). Further exemplary PTDs include but are not limited to, KKRRQRRR (SEQ ID NO:32), RKKRRQRRR (SEQ ID NO:38); an arginine homopolymer of from 3 arginine residues to 50 arginine residues.

Genetic constructs to encode TAL effector-based proteins or a recombinant “frataxin-protein transduction domain” protein can be made using either conventional gene synthesis or modular assembly. A plasmid kit for assembling custom TAL effector constructs is available through the public, not-for-profit repository by AddGene. Webpages providing access to public software, protocols, and other resources for TAL effector-DNA targeting applications include the “TAL Effector-Nucleotide Targeter” (http://boglabx.plpiastate.edu/TALENT/) and “taleffectors.com”. In an embodiment, the TAL effector-based recombinant protein of the present invention is made in accordance with the assembly protocol of Zhang et al., (2011).

In one aspect, the present TAL effector-based recombinant proteins of the present invention may be used to increase/induce expression of the frataxin nucleic acid and the frataxin protein in cells. As used herein, the expression “increasing” in “increasing the expression of frataxin in a cell” is meant to include circumstances where, in the absence of a TAL effector-based recombinant protein of the present invention, the frataxin protein is not expressed at all in said cell and where the cell already expresses a certain amount of frataxin protein. It comprises increasing/enhancing expression of frataxin in cells expressing no frataxin, a normal level or abnormal/lower level of frataxin as compared to normal conditions.

In an embodiment, the TAL effector-based recombinant proteins of the present invention may be used to increase transcription of the frataxin promoter in cells from a subject in need thereof. Non-limiting examples of a subject in need thereof include a subject having cells showing a reduced level of frataxin expression or activity as compared to cells from a normal subject. In an embodiment, the subject in need thereof is a subject having an abnormal number of trinucleotide repeats in intron 1 of the frataxin gene. In an embodiment, said number of trinucleotide repeats is 35 or more, 65 or more, 75 or more, 85 or more, 100 or more, 110 or more, 125 or more, 150 or more, 175 or more, 200 or more, 225 or more, 250 or more, 300 or more, 350 or more, 500 or more. In an embodiment, said subject in need thereof suffers from Friedreich ataxia. In an embodiment, the subject is a mammal, preferably, a human.

In a further embodiment, the recombinant “frataxin-protein transduction domain” protein may be used to increase may be used to increase levels of frataxin protein or a functional fragment and/or derivative thereof in cells, for example in cells from a subject in need thereof.

As used herein, “a subject in need thereof” is a subject, which may benefit from an increased expression of the frataxin protein or of increased levels of the frataxin protein.

In an embodiment, the present invention relates to a method of increasing frataxin expression in a subject in need thereof comprising administering an effective amount of a TAL effector-based recombinant protein of the present invention. In an embodiment, the recombinant protein is specifically formulated for crossing the plasma membrane and reaching the nucleus. In an embodiment, the present invention provides a composition comprising a TAL effector based recombinant protein of the present invention together with a pharmaceutically acceptable carrier.

In an embodiment, the present invention relates to a method of increasing frataxin (or a functional derivative and/or fragment thereof) levels in a subject in need thereof, comprising administering an effective amount of the recombinant “frataxin-protein transduction domain” protein of the present invention. In an embodiment, the present invention provides a composition comprising a recombinant “frataxin-protein transduction domain” protein of the present invention together with a pharmaceutically acceptable carrier.

Optimization of Codon Degeneracy

Because TAL effectors are expressed from bacterial pathogen infecting plants, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing TAL effector-based recombinant protein of the present invention.

Accordingly, the following codon chart (Table 2) may be used, in a site-directed mutagenic scheme, to produce nucleic acids encoding the same or slightly different amino acid sequences of a given nucleic acid:

TABLE 2 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Sequence Similarity

“Homology” and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is “homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term ‘homologous’ does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. For the sake of brevity, the units (e.g., 66, 67 . . . 81, 82, . . . 91, 92% . . . ) have not systematically been recited but are considered, nevertheless, within the scope of the present invention.

Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 2010, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 2010, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

In another aspect, the invention further provides a nucleic acid encoding the above-mentioned TAL effector-based recombinant protein or recombinant “frataxin-protein transduction domain” protein. The invention also provides a vector comprising the above-mentioned nucleic acid. In an embodiment, the vector further comprises a transcriptional regulatory element operably-linked to the above-mentioned nucleic acid. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, “operably-linked” DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory element” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, which induce or control transcription of protein coding sequences with which they are operably-linked.

In yet another aspect, the present invention provides a cell (e.g., a host cell) comprising the above-mentioned nucleic acid and/or vector. The invention further provides a recombinant expression system, vectors and host cells, such as those described above, for the expression/production of a recombinant protein, using for example culture media, production, isolation and purification methods well known in the art.

In another aspect, the present invention provides a composition (e.g., a pharmaceutical composition) comprising the above-mentioned TAL effector-based recombinant protein or recombinant “frataxin-protein transduction domain” protein. In an embodiment, the composition further comprises one or more pharmaceutically acceptable carriers, excipients, and/or diluents.

As used herein, “pharmaceutically acceptable” (or “biologically acceptable”) refers to materials characterized by the absence of (or limited) toxic or adverse biological effects in vivo. It refers to those compounds, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the biological fluids and/or tissues and/or organs of a subject (e.g., human, animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The present invention further provides a kit or package comprising the above-mentioned TAL effector-based recombinant protein or recombinant “frataxin-protein transduction domain” protein, or composition, together with instructions for increasing frataxin expression or levels in a cell or for treatment of Friedreich ataxia in a subject.

The present invention is illustrated in further details by the following non-limiting examples.

Example 1 Tal-Effector Based Recombinant Proteins Efficiently Promote Fratexin Expression in Cells

The method of Zhang et al. (2011) using a reporter plasmid has been used to design and generate several TAL effector based recombinant proteins, which are specific for various portions of the human frataxin promoter sequence. The mCherry reporter plasmid sold by AddGene™ inc. was not functional. Therefore, Applicants made their own reporter plasmid: pCR3.1-frataxin-promoter-miniCMV-mCherry, (see FIG. 21 and FIG. 22, and SEQ ID NO:43). Table 3 summarizes the nucleotide sequences, which have been targeted and the corresponding repeat-variable di-residue (RVD) (Cermak et al.) of the TAL effector based recombinant protein according to embodiments of the present invention.

TABLE 3 TAL-effector based Targeted recombinant sequence Targeted protein (NM_000144.4) sequence RVD repeats  1   5-18 tcccttgggtcagg HD HD HD NG NG NN NN NN NG HD NI NG NN; (SEQ ID NO: 1) (SEQ ID NO: 2)  2  21-34 tcctggttgcactc HD HD NG NN NN NG NG NN HD NI HD NG HD; (SEQ ID NO: 3) (SEQ ID NO: 4)  3  24-37 tggttgcactccgt NN NN NG NG NN HD NI HD NG ND HD NN NG; (SEQ ID NO: 5) (SEQ ID NO: 6)  4  37-50 tgctttgcacaaag NN HD NG NG NG NN HD NI HD NI NI NI NN; (SEQ ID NO: 7) (SEQ ID NO: 8)  5  73-86 tgccgaatagtgc NN HD NI HD NN NI NI NG NI NN NG NN HD; (SEQ ID NO: 9) (SEQ ID NO: 10)  6  81-94 tagtgctaagctgg NI NN NG NN HD NG NI NI NN HD NG NN; (SEQ ID NO: 11) (SEQ ID NO: 12)  7  92-105 tgggaagttcttcc NN NN NN NI NI NN NG NG HD NG NG HD HD; (SEQ ID NO: 13) (SEQ ID NO: 14)  8 103-116 tcctgaggtctaac HD HD NG NN NI NN NN NG HD NG NI NI; (SEQ ID NO: 15) (SEQ ID NO: 16)  9 106-119 tgaggtctaacctc NN NI NN NN NG HD NG NI NI HD HD NG; (SEQ ID NO: 17) (SEQ ID NO: 18) 10 124-137 tgctcccccacaga NN HD NG HD HD HD HD HD NI HD NI NN NI; (SEQ ID NO: 19) (SEQ ID NO: 20) 11 155-168 tggccaccaggggt NN NN HD HD NI HD HD HD NI NN NN NN NN NG; (SEQ ID NO: 21) (SEQ ID NO: 22) 12 168-181 tcgccgcagcaccc HD NN HD HD NN HD NI NN HD NI HD HD HD (SEQ ID NO: 23) (SEQ ID NO: 24)

PCR3.1 expression plasmids containing a gene coding for various TAL effector-based proteins under the EF1-α promoter have been produced (FIG. 2). However, in these plasmids, the TALE domain has been fused with a VP64 (SEQ ID NO:26) sequence (TALE-VP64) to induce the expression of a downstream gene following the attachment of the TALE-VP64 protein. Moreover, the TALE-VP64 gene was also fused with the gene coding for the EGFP reporter protein. A sequence coding for a 2A peptide (amino acid sequence GDVEENPGP (SEQ IS NO: 39) has been inserted between VP64 (SEQ ID NO:26) and EGFP (SEQ ID NO:54 to produce the vector pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE. Following, the transfection of this plasmid in cells, there was the production of a single mRNA by transcription. However, 2 separated proteins (i.e., the TALE-VP64 and the EGFP) were produced during transcription because of the presence of the 2A peptide.

In order to determine which TALE-VP64 proteins were able to induce the expression of the frataxin gene a fluorescence-based reporter construct was prepared. The proximal frataxin promoter (SEQ ID NO: 88, Lie et al., 2010) was inserted in a reporter plasmid 3′ of a minimal CMV promoter and of a mCherry reporter gene (pCR3.1-frataxin-promoter-miniCMV-mCherry, FIG. 3). The frataxin promoter sequence present in this plasmid is illustrated in FIG. 4.

Expression in 293FT Cells

Transfection:

mCherry reporter activation was tested by co-transfecting 293FT cells with plasmids carrying TALEs and mCherry reporters. 293FT cells were seeded into 24 plates the day before transfection at densities of 0.8×10⁴ cells/well. Approximately 24 h after initial seeding, cells were transfected using Lipofectamine™ 2000 (Invitrogen). We used 500 ng of TALE and 30 ng of reporter plasmids per well. Transfection experiments were performed according to manufacturer's recommended protocol.

Flow cytometry: mCherry reporter activation was assayed by flow cytometry. Cells were trypsinized from their culturing plates about 18 h after transfection and resuspended in 200 μl of media for flow cytometry analysis. At least 10,000 events were analyzed for each transfection sample. The fold induction of mCherry reporter gene by TALEs was determined by flow cytometry analysis of mCherry expression in transfected 293FT cells, and calculated as the ratio of the total mCherry fluorescence intensity of cells from transfections with and without the specified TALE. All fold-induction values were normalized to the expression level of TALE as determined by the total GFP fluorescence for each transfection.

When human cells were transfected with pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE, only green fluorescence was detected (FIG. 5). The presence of green fluorescence confirmed the expression of the EGFP protein and thus indirectly confirmed the expression of the TALE-VP64 protein. In addition, when the pCR3.1-frataxin-promoter-miniCMV-mCherry construct was initially transfected in human cells alone at 30 ng/ml, very few cells expressed the red fluorescence (FIG. 5). However, when human cells were co-transfected with vectors (pLenti-EF1α-TALE-VP64-2A-EGFP-WPRE and pCR3.1-frataxin-promoter-miniCMV-mCherry) both green fluorescence and various amounts of red fluorescence were detected confirming the activity of the TAL effector-based recombinant proteins (FIGS. 6 and 7). This indicated that the TALE-VP64 constructs were attaching to the frataxin promoter sequence inducing the expression of the mCherry reporter gene. FIG. 8 summarizes the results. The more active TAL effector based recombinant proteins of the present invention (#6, 7 and 8) are inducing the expression of mCherry (red fluorescence) in a higher proportion of the green fluorescence.

Thus, the TALE-VP64 proteins are able to attached to the frataxin promoter and drive the expression of a gene placed downstream of this promoter. These TALE-VP64 proteins may be used to increase the expression of the frataxin gene in a subject's cells. The increased expression of the frataxin will permit to reduce or prevent the symptoms associated with Friedreich ataxia.

Example 2 Tale_(Frat/VP16) Significantly Increases the Expression of the Human Frataxin Gene

A plasmid coding for TALE_(Frat/VP64)#8 (FIG. 9) was nucleofected in normal fibroblasts. Using quantitative RT-PCR, Applicants have confirmed in 3 independent experiments that the expression of the frataxin mRNA (relative to GAPDH mRNA) in human cells was doubled or triple by TALE_(Frat/VP64)#8 when results were normalized with cells transfected with EGFP or non-transfected cells (Table 4). Two to 3 independent experiments per conditions were performed.

TABLE 4 Q-RT-PCR results Normalized relative to non- Normalized relative to EGFP- Tale # transfected cells transfected cells 6 63-140%  73-118% 7 94-127% 110-148% 8 165-313%  192-214%

Applicants have also shown that TALE #8 also increased by 2 fold the frataxin mRNA in fibroblasts from a patient suffering from Friedreich ataxia. Indeed, nucleofection of pCR3.1 TALE_(frat/VP64)#8 significantly increased the frataxin mRNA compared with a control not nucleofected (CON) or a control nucleofected with a plasmid coding for GFP (FIG. 10). The frataxin mRNA was amplified by PCR (using primers defined in SEQ ID NOs 90 and 91) and normalized with 3 different internal controls (HPTR1 (SEQ ID NOs:92 and 93), GAPDH (SEQ ID NOs: 94 and 95) and 18S rRNA (SEQ ID NOs: 96 and 97), see also Table 5 below). The Friedreich fibroblasts used for this experiment were obtained from Coriell Institute for medical (GM 04078) and have 541 and 420 repeats on intron 1 of each allele of the gene respectively.

TABLE 5  Primer sequences used for qRT-PCR Size of T Gene amplicon annealing Primer sequences Symbol Description GenBank (pb) (° C.) 5′→3′ S/AS FXN Homo sapiens frataxin (FXN), NM_000144 106 57 AAGCCATACACGTTTGAGGACTA/ nuclear gene encoding TTGGCGTCTGCTTGTTGATCA miotchondrial protein, region present in the 3 transcripts Hprt Homo sapiens hypoxanthine NM_000194 157 57 AGTTCTGTGGCCATCTGCTTAGTAG/ phosphoribosyltransferase 1 AAACAACAATCCGCCCAAAGG GAPDH Homo sapiens glyceraldehyde- NM_002046 194 57 GGCTCTCCAGAACATCATCCCT/ 3-phosphate dehydrogenase ACGCCTGCTTCACCACCTTCTT

For the 18S ribosomal RNA (NR_003286) primers: acggaccagagcgaaagcatt and tccgtcaattcctttagtttcagct (SEQ ID NO: 96 and SEQ ID NO:97), were used.

Results obtained at the mRNA level were also confirmed at the protein level. TALE #8 also increased by almost 2 fold the frataxin protein in fibroblasts from the same Friedreich patient (FIG. 11). Nucleofection of PCR 3.1 TALE_(frat/VP64) #8 significantly increased the frataxin protein compared to a non-nucleofected control (CON) or with a control nucleofected with a pCR3.1 plasmid coding for eGFP (GFP). Frataxin protein expression was normalized using β-actin as an internal standard. The mAb used to detect frataxin was #18A5DB1 from Mitosciences. Such an increase would be in the therapeutic range (i.e., 50% of normal frataxin level) for many patients.

Example 3 Increased Expression of Frataxin mRNA and Protein in Rescued YG8 Fibroblasts

The rescued YG8 (YG8R) mouse model has 2 null mouse frataxin genes but contains a human frataxin transgene (with its human promoter) obtained from a FRDA patient. This human transgene contains 230 GAA repeats in intron 1 and thus a reduced amount of human frataxin is produced leading to the development of FRDA symptoms in this mouse model. Applicants have shown that the transfection of TALE_(frat/VP64) #8 plasmid in YG8R fibroblasts also increases frataxin mRNA (by about 1.4 to 1.9 fold) and protein (by about 1.5 fold) (FIG. 12).

As shown in FIG. 12, Applicants have analyzed the effect of PCR3.1 TALE 8 on mRNA and protein levels of frataxin as follows: YG8 cells were cultured at near of 80% confluency in a T-75 flask (medium culture: DMEM High glucose, 10% bovine serum containing non-essential amino acids (1×) and pen-strep (1λ)), then the cells were trypsinized and divided equally in six tubes (15 ml sized) and centrifuged at low speed (1200×g) 5 minutes. The supernatant was discarded and cells were resuspended in 5 ml HBSS in each tube and then centrifuged as previously. At the end of centrifugation, the pellet (cells) was kept at room temperature. To each tube containing only the cells pellet was added a mix of nucleofection solution (VPD-1001)/plasmid DNA (100 μl/10 μg plasmid pCR3.1 TALE 8), cells were resuspended by pipetting up and down and transferred in a cuvette and placed inside of the Nucleofector device II™ apparatus (Amaxa biosystem)) set at the program P-022 and then nucleofected. Then, the nucleofected cells (from each nucleofection) were seeded in a well of a 6-well plate and maintained in culture overnight at 37° C. in a cells incubator. Several changes of culture medium (in the same culture medium described above) were done to remove cellular debris and culture was performed at 37° C. in an incubator during 50 to 60 hours before mRNA or protein were extracted and analysed by qRT-PCR or Western blot to evaluate Frataxin expression. Table 6 below presents the primer sequences used for qRT-PCR analysis in Y8G cells.

TABLE 6 Primers used for qRT-PCR analysis in Y8G cells following nucleofection of plasmid pCR3.1 TALE no.8. Mm Hprt1 Mus musculus hypoxanthine NM_013556 106 57 CAGGACTGAAAGACTTGCTCGAGAT/ guanine phosphoribosyl CAGCAGGTCAGCAAAGAACTTATA transferase 1 GC (SEQ ID NOs: 98 and 99) Mm Mus musculus NM_008084 123 57 ACGGGAAGCTCACTGGCATGG/ATG GAPDH glyceraldehyde-3-phosphate CCTGCTTCACCACCTTCTTG (SEQ dehydrogenase ID NOs: 100 and 101) Mm 18S 18S ribosomal RNA (Rn18s), NR_003278 119 57 TGGATACCGCAGCTAGGAATAATG/ ribosomal RNA TCACCTCTAGCGGCGCAATAC (SEQ ID NOs: 102 and 103) Mm ADNg Mus musculus chromosome 3 NT_039239 209 57 CACCCCTTAAGAGACCCATGTT/CC genomic contig, strain CTGCAGAGACCTTAGAAAAC (SEQ C57BL/6J (HSD3B1 intron) ID NOs: 104 and 105) Hs FXN Homo sapiens frataxin (FXN), NM_000144 106 57 AAGCCATACACGTTTGAGGACTA/TT nuclear gene encoding GGCGTCTGCTTGTTGATCA (SEQ ID mitochondrial protein, région  NOs: 90 and 91) commune aux 3 transcrits

For Frataxin protein expression analysis by Western blot, the same human antibodies described earlier were used since as indicated above, the human frataxin gene was introduced into the mouse genome. Furthermore, the human monoclonal β-actin antibody (Sigma) was also able to recognize mouse β-actin.

Example 4 Production And Purification of a 6×HIS-CPP-Tale_(Frat/VP64) Protein

Applicants have constructed 2 plasmids (6×His-Tat-TALEFratNP64 and 6×His-Pep1-TALEFrat/VP64) in pET-16B (Novagen inc.) coding for a TALE_(frat/VP64) protein fused with a cell penetrating peptide (CPP) and with a 6×His flag to permit the production of these recombinant proteins in E. coli (BL21) and their purification on a nickel affinity column. FIG. 13 shows the amino acid sequences of the proteins coded by these plasmids.

These plasmids permit the production of the recombinant protein in E. coli (BL21) following induction with 1 mM IPTG (FIG. 14A). The 6×His-Tat-TALE_(frat/VP64) has been purified on a nickel affinity column (FIG. 14B). The recombinant protein has been detected in Western blot with an anti-6×His mAb (Qiagen #34660) (FIG. 15A) and with a polyclonal anti-VP16 mAb (ab4809, Abcam) (FIG. 15B).

Production and Purification of 6×HIS-CPP-TALE frataxin/VP64 Protein:

The recombinant plasmids TALE No. 8 (6×HIS-TAT or PEP1) in the prokaryotic expression vector pet-16b (Novagen) have been transformed in BL21 DE3 pLysS and grown overnight at 37° C. on an agar plate containing ampicillin (150 μg/ml, chloramphenicol (34 μg/ml). The next day, three to five colonies of each recombinant were seeded separately in 25 ml of LB containing ampicillin (150 μg/ml) and chloramphenicol (34 μg/ml) and were grown under agitation at 37° C. The next day, a new culture was started by seeding 10 ml of the overnight culture in 200 ml of LB medium containing only ampicillin at 150 μg/ml. When the culture reached Absorbance (A600 nm) near 0.5-0.7, induction with 1 mM IPTG was done and the culture is kept at 37° C. during four hours under vigorous shaking conditions at 37° C. After, the culture (for each recombinant) was centrifuged at 4000 rpm 15 minutes and pellets were kept frozen at −80° C. until lysis. Lysis was performed using the lysis solution (10 ml) CelLytic B™ (Sigma) diluted at 1/10 in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH8.0. Benzonase (25 U/ml) and protease inhibitors were preferably added and vigorous shaking of the lysate was performed at room temperature during 20 minutes. Then, centrifugation was performed at 16000×g during 20 minutes and the supernatant (10 ml) (containing the soluble recombinant TALE 8) was passed through a column (NI-NTA Superflow, size 25 ml) (Qiagen). The column was washed with three volumes (30 ml) of washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole pH8.0) and elution was performed with 10 ml of 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole pH8.0. The elution fraction (2 ml) was collected in the first and second tubes and were passed on a Zeba™ desalting column (Thermo Fisher Pierce) in PBS containing L-arginine 50 mM and L-Glutamine 50 mM to remove imidazole and keep soluble the recombinant protein.

Example 5 Cell Lines and Animal Models of Friedreich Ataxia

Cell Line Models

For research on Friedreich ataxia, there is a collection of lymphoblasts and fibroblasts, from FRDA patients, their parents and their siblings available at the National Institutes of General Medical Sciences Human Genetic Cell Repository (Coriell Institute, New Jersey) (http://www.coriell.org/nigms). These cells may be used for validating that TALEs can increase the expression of frataxin in cells of Friedreich ataxia patients. In this collection, there are FXN alleles with different repeat numbers.

Animal Models

Several animal models have been made. The first one is a heterozygous knockout mouse (KO/Normal FXN). This mouse shows reduced (50%) frataxin levels, and exhibits sporadic heart iron deposits after dietary iron load (Santos et al. 2003). The homozygous KO mouse (KO/KO) is not viable and dies during embryonic development. Further, a KI (knock-in) mouse was developed with a 230 GAA repeat. The homozygous KIKI mice have frataxin protein at about 75% of the wild type mice (Miranda et al. 2002). The KI and heterozygous KO mice have been bred together to produce heterozygous KIKO. These mice expressed only 25-36% of the WT frataxin protein at 12 months of age (Miranda et al. 2002). Conditional KO/KO mouse models have been produced with absence of frataxin in muscle heart, brain or pancreas (Puccio et al. 2001), (Ristow et al. 2003). Depending on the tissue in which the gene is knocked out, these mice developed cardiac hypertrophy, large sensory neuron dysfunction or diabetes due to reactive oxygen species increase, growth arrest and apoptosis in pancreatic beta cells. In these conditional KO with a Cre under a MCK promoter, there is in the heart a progressive increase of two important mitochondrial ATP dependent proteases (Lon and CIpP), a simultaneous and significant progressive loss (loss of 80%) of mitochondrial Fe—S proteins (aconitase and ferrochelatase) and of SDHA and ND6 respiratory chain subunits (Guillon et al. 2009). These changes are detectable at 3 weeks and are very pronounced at 5 weeks.

Other researchers have developed transgenic mice containing a YAC containing a human FXN gene with either 190 or 280 GAA repeats, respectively designated as YG22 and YG8. These mice showed an intergenerational and age-related somatic instability of the repeats, with the most prominent expansions occurring specifically in the cerebellum (as seen in FRDA patients) and in the dorsal root ganglia (Al-Mandawi et al. 2006, Clark et al. 2007). By crossbreeding the FXN YAC transgenic mice with heterozygous FXN KO mice obtained from Puccio and colleagues, the Pook laboratory has further shown that both transgenes are able to successfully rescue homozygous FXN knockout (KO/KO) embryonic lethality and that the rescue mice (called YG22 rescued and YG8 rescued) express no mouse frataxin and only a low level of human frataxin (because they have only one human FNX gene, which has a 190 or 280 GAA repeat) and thus they exhibit an FRDA-like phenotype (Al-Mandawi et al. 2006). These rescued mice (particularly the YG8 rescued mice) exhibited decreased frataxin mRNA and protein in affected organs, decreased coordination ability (as determined by rotarod testing), decreased aconitase activity, oxidative stress (decreases CuZnSOD and MnSOD in muscles, cerebellum and heart), histopathology in DRG large sensory neurons, iron accumulation in the heart, marked reduction of activity starting at 6 months and increased weight (Al-Mandawi et al. 2006, Clark et al. 2007). Therefore, this is an excellent FRDA mouse model in which to investigate the potential therapeutic effects of the TALEs targeting the human frataxin promoter.

Other researchers have produced a FXN-EGFP reporter mouse, i.e., a transgenic mouse with a natural FXN promoter controlling the expression of EGFP (Grant et al. 2006, Sarsero et al. 2003, Sarsero et al. 2005). This transgenic model is useful to screen for compounds able to increase the activity of the FXN promoter.

Interestingly, a transgenic mouse over-expressing frataxin has been produced (Miranda et al. 2004). This transgenic mouse does not develop any pathology. This is an important result because this clearly shows that if frataxin protein is increased over the normal level by an eventual therapy in FRDA patients, the over abundance of frataxin will not produce adverse effects.

Example 6 A Recombinant Frataxin Protein Fused with a Protein Transduction Domain Prevents the Death of Fibroblasts with a Knock Out Frataxin Gene

Construction of the Frataxin Expression Vector

A bacterial expression vector was produced. The human frataxin cDNA was obtained from Origene inc. (Rockville, Md.) and cloned in the pET-16b (Novagen inc., Merck inc., Darmstadt, Germany) vector under a Lac-operon inducible by Isopropyl β-D-1-thiogalactopyranoside (IPTG, BioBasic inc., Amherst, N.Y.). Various versions of the expression vector were done with the frataxin cDNA fused or not at its 5′ end with a sequence coding for a protein transduction domain (PTD), i.e., either Tat or Pep-1 (Morris et al. 2001, Vives et al. 2003, Morris, Heitz and Divita 2002). However, to facilitate the protein purification this PTD was preceded by a sequence coding for a 6×His tag. Some vector variants also included a V5-tag (Invitrogen inc., Carlsbad, Calif.) at the 3′ end.

Production and Purification of the Recombinant Frataxin

The various recombinant frataxin proteins (6×His-frataxin, 6×His-Tat-frataxin, 6×His-Pep-1-frataxin, 6×His-frataxin-V5, 6×His-Tat-frataxin-V5, 6×His-Pep-1-frataxin-V5) were produced in E. coli transformed with the pET-16b vector. Thus, all expression vectors included a sequence coding for a 6×His tag to permit the purification of the proteins on a nickel affinity column. The bacteria were grown in LB medium at 37° C. until a DO₆₀₀ between 0.5 and 1.0. The protein production was induced by adding IPTG to a final concentration of 0.5 mM. The bacteria were lysed 4 hours later with Cellytic B™ (Sigma inc., St-Louis, Mo.). The bacteria were centrifuged and the supernatant was flown through a Nickel affinity column (Qiagen inc., Valencia, Calif.). The column was first washed with the NPI-10 buffer. The frataxin protein was eluted with 100 mM imidazole. The purity of the protein was confirmed by gel electrophoresis followed by Coomassie blue staining (FIG. 17) or by western blot with a mAb against frataxin (Mito Science inc., Eugene, Oreg.).

Culture of the Conditional Knockout Fibroblasts

A murine cellular model for FRDA was used for several of our experiments (Calmels et al. 2009). This fibroblast line carries a null frataxin gene and a conditional frataxin allele. These fibroblasts were grown in DMEM media (Sigma, Saint Louis, Mo.) with 10% fetal calf serum. When these cells are transfected with an EGFP-Cre recombinase gene under a CMV promoter (AddGene inc., Cambridge, Mass.), this creates a cell model depleted of endogenous frataxin. The complete absence of murine frataxin in these fibroblasts inhibits cell division and leads to cell death.

Western Blot for the V5-Tag

To detect the V5-tag in western blot, the membrane was incubated with an anti-V5 mAb (Invitrogen inc.). The presence of the mAb was detected with an anti-mouse coupled with horseradish peroxidase (HRP) followed by chemiluminescence detection.

Transfection of 293FT cells with plasmids coding for frataxin-V5 or 6×His-frataxin-V5

Normally, the frataxin protein is produced in the cytoplasm as a pro-frataxin containing 210 amino acids. This pro-protein is then imported in the mitochondria where the N terminal is truncated twice to produce an intermediary and a mature frataxin. To verify whether the presence of the 6×His tag and of a PTD in the recombinant frataxin could prevent its maturation, 293FT cells were transfected with plasmids coding either for frataxin fused with the V5 tag (pCR3.1-Frataxin-V5) or for 6×His-Pep-1-frataxin fused with the V5 tag (pCR3.1-6×His-Pep-1-Frataxin-V5). The proteins were extracted from the cells 36 hours later. A V5 mAb was used to detect the recombinant proteins in a western blot. Three isoforms of the frataxin proteins were detected: PP: pro-protein, PI: intermediary protein, PM: mature protein (see FIG. 16). When the cells were transfected with the plasmid coding for Frataxin-V5, some pro-proteins and intermediary proteins were detected but most of the proteins were already mature. When the cells were transfected with the plasmid coding for the 6×His-Pep-1-Frataxin-V5, most of the proteins were in the pro-protein isoforms but some intermediary proteins and mature proteins were also detected. This indicates that although the presence of a PTD may delay the maturation of the frataxin, the protein was nevertheless able to mature.

The frataxin gene in the conditional knock-out fibroblasts was knock-out by transfecting these cells with a Cre-recombinase-EGFP plasmid. The green fluorescent cells, i.e., transfected cells, were selected by FACS and placed back in culture. These cells were thus not able to produce frataxin. Some cells were cultured without any frataxin supplement in the culture medium or with 6×His-frataxin. All these cells died in less than one week (FIGS. 18 B and C). However, when Tat-frataxin or Pep-1-frataxin was added to the culture medium, the frataxin-KO fibroblasts survived and proliferated (FIGS. 18 D and E). This indicates that the recombinant proteins coupled with a PTD were not only able to enter into the fibroblasts but that they also entered in the mitochondria and were able to replace the missing frataxin protein leading to cell survival.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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1-20. (canceled)
 21. A method for treating Friedreich ataxia in a subject comprising administering to said subject a transcription activator-like (TAL) effector-based recombinant protein or a nucleic acid encoding said recombinant protein, said recombinant protein comprising: i) a TAL effector domain comprising a repeat variable domain (RVD) comprising at least 10 tandem repeat monomers and a half monomer at the C-terminal end of the RVD, each monomer corresponding to an HD, NG, NN, NI, NK or NS module consisting of the amino acid sequence set forth in SEQ ID NO: 65 and the half monomer being a truncated monomer consisting of the first 13 contiguous amino acids of SEQ ID NO: 65, ii) a nuclear localization signal; and iii) a transcription activation domain, wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88 of a mammalian frataxin gene comprising at least 75 repeats of GAA trinucleotides in intron 1 thereof, and wherein said recombinant protein increases frataxin expression in said cell.
 22. A method for increasing frataxin expression in a cell comprising a frataxin gene comprising at least 75 repeats of GAA trinucleotides in intron 1 thereof, the method comprising transducing said cell with a transcription activator-like (TAL) effector-based recombinant protein or nucleic acid encoding said recombinant protein, said recombinant protein comprising: i) a TAL effector domain comprising a repeat variable domain (RVD) comprising at least 10 tandem repeat monomers and a half monomer at the C-terminal end of the RVD, each monomer corresponding to an HD, NG, NN, NI, NK or NS module consisting of the amino acid sequence set forth in SEQ ID NO: 65 and the half monomer being a truncated monomer consisting of the first 13 contiguous amino acids of SEQ ID NO: 65, ii) a nuclear localization signal; and iii) a transcription activation domain, wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88 of a mammalian frataxin gene in said cell, thereby increasing frataxin expression in said cell.
 23. The method of claim 21, wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 24. The method of claim 21, wherein said RVD binds to: i) Nucleotides 5-18 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; ii) Nucleotides 21-34 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; iii) Nucleotides 24-37 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; iv) Nucleotides 37-50 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; v) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; vi) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or vii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or viii) Nucleotides 106-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:
 88. 25. The method of claim 23, wherein said RVD binds to: i) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; ii) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or iii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:
 88. 26. The method of claim 21, wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules: i) HD-HD-HD-NG-NG-NN-NN-NN-NG-HD-NI-NG-NN; ii) HD-HD-NG-NN-NN-NG-NG-NN-HD-NI-HD-NG-HD; iii) NN-NN-NG-NG-NN-HD-NI-HD-NG-ND-HD-NN-NG; iv) NN-HD-NG-NG-NG-NN-HD-NI-HD-NI-NI-NI-NN; v) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN; vi) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; vii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI; or viii) NN-NI-NN-NN-NG-HD-NG-NI-NI-HD-HD-NG; wherein the C-terminal module in said configuration of modules corresponds to the half monomer, wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63, and wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:
 88. 27. The method of claim 23, wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules: i) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN; ii) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; or iii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI. wherein the C-terminal module in said configuration of modules corresponds to the half monomer, wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63; and wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:
 88. 28. The method of claim 21, wherein said transcription activation domain comprises at least one VP16 synthetic transcription activation domain.
 29. The method of claim 21, wherein said transcription activation domain comprises at least one VP64 synthetic transcription activation domain.
 30. The method of claim 21, wherein said nuclear localization signal is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen.
 31. The method of claim 22, wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 32. The method of claim 22, wherein said RVD binds to: i) Nucleotides 5-18 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; ii) Nucleotides 21-34 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; iii) Nucleotides 24-37 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; iv) Nucleotides 37-50 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; v) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; vi) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or vii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or viii) Nucleotides 106-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 33. The method of claim 31, wherein said RVD binds to: i) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; ii) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or iii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 34. The method of claim 22, wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules: ix) HD-HD-HD-NG-NG-NN-NN-N N-NG-HD-NI-NG-NN; x) HD-HD-NG-NN-NN-NG-NG-NN-HD-NI-HD-NG-HD; xi) NN-NN-NG-NG-NN-HD-NI-HD-NG-ND-HD-NN-NG; xii) NN-HD-NG-NG-NG-NN-HD-NI-HD-NI-NI-NI-NN; xiii) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN; xiv) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; xv) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI; or xvi) NN-NI-NN-NN-NG-HD-NG-NI-NI-HD-HD-NG; wherein the C-terminal module in said configuration of modules corresponds to the half monomer, wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63, and wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 35. The method of claim 31, wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules: i) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN; ii) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; or iii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI. wherein the C-terminal module in said configuration of modules corresponds to the half monomer, wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63; and wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.
 36. The method of claim 22, wherein said transcription activation domain comprises at least one VP16 synthetic transcription activation domain.
 37. The method of claim 22, wherein said transcription activation domain comprises at least one VP64 synthetic transcription activation domain.
 38. The method of claim 22, wherein said nuclear localization signal is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen. 